Recombining nucleic acids has useful applications such as finding sequences which produce products having improved or desired characteristics. In particular, it is useful to develop a method for the production of mutant proteins which method allowed for the development of large libraries of mutant nucleic acid sequences which were easily searched.
A variety of in vitro DNA recombination methods exist. Examples include those described in U.S. Pat. No. 5,605,793 and in U.S. Pat. No. 5,965,408. Generally, recombination methods depend on a step of making fragments, and a step of recombining the fragments. For example, U.S. Pat. No. 5,605,793 generally relies on fragmentation of double stranded DNA molecules by DNase I. U.S. Pat. No. 5,965,408 generally relies on the annealing of relatively short random primers to target genes and extending them with DNA polymerase. Each of these disclosures relies on polymerase chain reaction (PCR)-like thermocycling of fragments in the presence of DNA polymerase to recombine the fragments.
Generally, existing methods generate DNA fragments with functional 3′ ends, which can be readily extended in the presence of complementary sequences. Thus, in one of the examples described above, a consequence of this feature is that fragments in the assembly or recombination step are independent from each other. The fragments can anneal to their complementary counterparts and get extended by DNA polymerase irrespectively of their position in the primary sequences of the gene—extension of different fragments occurs in parallel.
Although, a number of methods exist, it would generally be desirable to provide novel methods of recombining nucleic acids. Moreover, it would be desirable to provide a method of forming recombined nucleic acid molecules which took into account the position of a fragment relative to the primary or initial sequence from which it was fragmented. In particular, it would be desirable to provide methods of forming recombined nucleic acid molecules wherein a sequence was fragmented, and wherein the fragments were used as templates only, rather than as templates and primers for extension.